Sublytic Of C5b-9 Induced Thy-1 Nephritis Gmcs Mechanism Of Apoptosis

PartⅠAnalysis of the relationship between complement C5b-9 complexes desposits in glomeruli of rats with Thy-1 N and apoptosisAIMS:(1)To reproduce rat model of human mesangial proliferative glomerulonephritis (MsPGN),namely,Thy-1 nephritis(Thy-1 N),and to investigate the pathologic changes of rats with Thy-1 N during different phases and the relationship between the deposition of C5b-9 complexes and apoptosis.METHODS:(1)Thy-1 N rats were reproduced with Thy-1 antibody(Thy-1 Ab)via intravenous injection.The rats were divided into 3 groups,namely,Thy-1 N group,CVF+Thy-1 N group and control group.The complement C5b-9 desposits in kiney of rats with Thy-1 N were observed by immunohistochemistry(IHC).The pathologic changes such as glomerular mesangial cells(GMCs)apoptosis,lysis and proliferation were examined by light microscope(LM),electron microscope(EM),and Tdt-mediated X-dUTP nick end labeling(TUNEL)at 40 min,3 h,24 h and 7 d after administration ofThy-1 Ab or normal serum.(2)Based on the formation of sublytic C5b-9 complexes,the GMCs were divided into 4 groups,namely,Thy-1 N group,CVF+Thy-1 N group and control group.The apoptosis of GMCs was evaluated by Flow cytometry after Annexin V-propidium iodide(PI)staining.RESULTS:(1)The deposition of complement C5b-9 complexes were observed in glomerular mesangium on the surface of GMCs at 40 min and 3 h after Thy-1 antibody injection and the C5b-9 distribution was investigated to be similar to that of Thy-1 antibody(data not shown).However,the lysis of the GMCs with C5b-9 complexes deposition was not shown at the same time. (2)Under LM,GMCs were shown to be lysis,and the numbers of glomerular cells were observed to be decreased at 24 h,then increased on 7 d.Under EM,glomerular GMCs were exhibited with apoptosis at 40 min,lysis at 24 h,proliferation and extracellular matrix(ECM)accumulation on 7 d after Thy-1 Ab administration. However,the number of apoptotic glomular cells in CVF+Thy-1 N group was markedly less than that in Thy-1 N group at 40 rain and 3 h.In addition,the numbers of TUNEL-positive cells in the glomeruli of Thy-1 N were shown to be significant increasing at 40 min and 3 h by TUNEL staining,and with the progess of Thy-1 N, TUNEL-positive cells decreased gradually.Otherwise,in CVF+Thy-1 N rats,the number of TUNEL-positive cells in the glomeruli significantly decreased,compared with Thy-1 N group.(3)The number of apoptotic GMCs in sublytic C5b-9 group was more than that in other groups at 3 h.CONCLUSIONS:GMCs apoptosis in early time of Thy-1 nephritis is related to the desposits of complement C5b-9 complexes. PartⅡGene expression profiles and up-regulation of IRF-1 in vivo and in vitroAIMS:(1)To detect expressive genes from renal tissue of rats with Thy-1 N and GMCs with sublytic C5b-9 stimulation at 40 rain and 3 h.(2)To ensure the mRNA and protein levels of IRF-1 for exploring the reason of GMCs apoptosis. METHODS:(1)The changes of gene expression profiles from renal tissue of rats with Thy-1 N and at 40 min and 3 h were detected by microarray technique.Then the up-regulated genes were analyzed in Batch Quary of Affymetrix and Genbank to find out their functions.(2)The gene of interferon regulatory factor-1(IRF-1)was chosen and its levels of mRNA and protein were measured by reverse transcription PCR (RT-PCR),real-time PCR,Western blotting and Immunohistochemistry.RESULTS: (1)Microarray analysis showed that,at 40 min and 3 h,the up-regulation of 667 genes and 173 genes were exhibited in Thy-1 N,while 536 genes and 486 genes were up-regulated in the cultured GMCs following sublytic C5b-9 attack.These differentially expressed genes were associated with apoptosis,signal transduction, enzyme,cytokine receptors,proliferation,and so on.(2)Among all differentially expressed genes,IRF-1,activating transcription factor 3(ATF3),connective tissue growth factor(CTGF),growth arrest and DNA-damage-inducible 45(Gadd 45),dual specificity phosphatase 1(MKP-1),heme oxygenase 1(HO-1),schlafen 3,enhancer binding protein(C/EBP),Cyclin L1 and V-jun sarcoma virus 17 oncogene homolog (avian)were found to be up-regulated both in vivo and in vitro at the same time.(3) The mRNA transcripts of IRF-1 were found to be significantly up-regulated at 40 min and 3 h compared to control by RT-PCR and real-time PCR,which was similar to changes in microarray experiments.And meanwhile,the IRF-1 protein from renal tissues in the Thy-1 N group was present at 3 h,but was absent at 40 min and in the control group,and IRF-1 protein from GMCs with sublytic C5b-9 treatment was markedly increased at 3 h and the distribution of IRF-1 protein was largely in the nuclei of GMCs.CONCLUSIONS:The pathological changes of Thy-1 N were correlation with the abnormal expression of many genes.Moreover,the up-regulated IRF-1 gene was co-expressed in both renal tissues of rats with Thy-1 N(in vivo)and the cultured GMCs induced by sublytic C5b-9(in vitro.). PartⅢThe role and mechanism of interferon regulatory factor-1(IRF-1)on apoptosis of glomerular mesangial cells (GMCs)induced by sublytic C5b-9AIMS:To explore the role and the mechanism of interferon regulatory factor-1 (IRF-1)on apoptosis of glomerular mesangial cells(GMCs)induced by sublytic C5b-9.METHODS:The coding sequence of the IRF-1 gene was obtained by reverse transcription-PCR(RT-PCR)with the designed primers and inserted into the BamHⅠ/HindⅢsites of pcDNA3.1/myc vector to construct pcDNA3.1/IRF-1-myc plasmid.Then the construction of pcDNA3.1/IRF-1-GFP were done by sub-clone way. The cultured rat GMCs was transfected with pcDNA3.1/myc,IRF-1 plasmid (pcDNA3.1/IRF-1-myc or pcDNA3.1/dnIRF-1-GFP)and dominant negative IRF-1 plasmid(pcDNA3.1/dnIRF-1-myc or pcDNA3.1/dnIRF-1-GFP)respectively,with or without sublytic C5b-9 attack,then the cells were divided into different groups.The level of IRF-1 expression was assessed by microscope fluorescent and Western blotting.The nuclear morphology of GMCs was observed with Hoechst 33342 staining,and sub-G1 DNA content and alteration in cell cycle were analyzed by Flow cytometry.The content of IFN-β,γin the cultured supernatant was detected by ELISA. The activaty of cysteine-dependent aspartate protease(Caspase)-3/7 in GMCs was evaluated by Caspases 3/7 substrate Z-DEVD-R110.RESULTS:(1)The expression plasmids(pcDNA3.1/IRF-1-myc and pcDNA3.1/IRF-1-GFP)were successfully constructed.(2)The cultured GMCs were transfected with expression vectors of pcDNA3.1/IRF-1 or pcDNA3.1/dnIRF-1,and the best transfection time was ensured to be 48 h.(3)In sublytic C5b-9-induced GMCs and pcDNA3.1/IRF-1-transfected GMCs,the typical apoptotic chromatin condensation and fragmentation were seen and the ratio of apoptotic cells was increased as well as cell cycle was arrested at G1 phase.In addition,the productions of IFN-β,γand the activatity of Caspase-3/7 were all enhanced.Moreover,the obvious increase above-mentioned was shown in pcDNA3.1/IRF-1-transfected GMCs with sublytic C5b-9 attack.In constract,the numbers of apoptotic GMCs,the G1-arrest,and the production of IFN-βas well as the activatity of Caspase-3/7 in response to sublytic C5b-9 attack were subdued mostly by dnIRF-1 through competing with IRF-1.However,no marked decrease on GMCs apoptosis induced by pcDNA3.1/IRF-1+ sublytic C5b-9 after neutralizing IFN-βand IFN-γwere shown.CONCLUSIONS:The GMCs apoptosis induced by sublytic C5b-9 attack was associated with up-regulation of IRF-1 via increasing Caspase 3/7 activity.